Background

The complement system plays an important role in many neurological disorders.

Complement modulation, including C3/C3a receptor signaling, shows promising therapeutic effects on cognition and neurodegeneration. Yet, the implications for this pathway in perioperative neurocognitive disorders (PND) are not well established. Here, we evaluated the possible role for C3/C3a receptor signaling after orthopedic surgery using an established mouse model of PND.

Methods

A stabilized tibial fracture surgery was performed in adult male C57BL/6 mice under general anesthesia and analgesia to induce PND-like behavior. Complement activation was assessed in the hippocampus and choroid plexus. Changes in hippocampal neuroinflammation, synapse numbers, choroidal blood-cerebrospinal fluid barrier (BCSFB) integrity, and hippocampal-dependent memory function were evaluated after surgery and treatment with a C3a receptor blocker.

Cognitive impairments are common problems especially amongst older surgical patients [1]. These neurological complications, termed as perioperative neurocognitive disorders (PND) [2], associate with poor functional recovery and increased mortality after major surgery [3]. Although the pathogenesis of PND remains unclear, preclinical studies suggest that surgery triggers acute systemic inflammation [4] followed by neuroinflammation [5–7] and synaptic dysfunction [8, 9], which appear to contribute to hippocampal-dependent cognitive deficits. Recent human studies describe similar pathological hallmarks after major surgery including biomarkers of systemic inflammation, neuroinflammation, and neuronal damage [10, 11]. Strategies aimed at modulating this immune response have shown promising effects in animal models; however, no effective strategies for the treatment and/or prevention of PND are available for clinical use yet.

The complement system is well-known to play an important role in innate immunity regulation [12]. Emerging evidence shows that the complement system also serves many pivotal functions in the central nervous system (CNS) [13, 14]. Under homeostatic conditions, complement pathways help eliminating cellular debris, apoptotic cells, and pathogens [13], as well as regulating synaptic pruning during brain development [15]. In contrast, abnormal activation of the complement system has been related to several CNS pathologies and neurodegenerative conditions [16, 17].

The central component of the complement system, C3, has been extensively investigated in the CNS [16–21]. C3a, a cleavage product of C3, binds to the G protein-coupled receptor named C3a receptor (C3aR) [22]. Both pharmacological blockade and genetic deficiency in C3aR have therapeutic effects in models of neuroinflammation, synapse loss, and cognitive dysfunction in rodents [17, 19, 20]. Herein, we hypothesize that orthopedic surgery induces C3/C3aR signaling activation in the CNS, thus contributing to PND pathogenesis. Further, we demonstrate that administration of C3a can trigger neuroinflammation whereas C3aR blockade provides therapeutic benefits that may inform about novel clinical trials.

Mice

All experiments were approved by Institutional Animal Care and Use Committee at Capital Medical University (Beijing, China) and performed under the regulations of Medical Research Center of Beijing Chao-Yang Hospital (Beijing, China). Twelve- to 14-week-old male C57BL/6 mice were purchased from Vital River Laboratory (Beijing, China). All mice were housed under a 12 h light/dark cycle with free access to food and water in the vivarium of Beijing Chao-Yang Hospital.

Experimental design

Study design. a Mice were randomly assigned to three groups: naive, sham, and surgery. Mice were sacrificed for tissues harvesting at 6 h, 1 day, and 3 days after surgery or sham. b Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + C3aR antagonist (C3aRa). C3aRa or vehicle was given 1 h prior to surgery or sham. Mice were sacrificed for tissues harvesting at 6 h and 1 day after surgery or sham. c Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + C3aR antagonist (C3aRa). 30 min after C3aRa or vehicle administration, mice were subjected to the training session for trace fear conditioning. Mice underwent surgery or sham 30 min after training. At 3 days after surgery or sham, context test was performed. d Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + recombinant mouse C3a (rmC3a). Mice were given intranasal rmC3a or vehicle at 24 h and 48 h after surgery or sham procedure. Mice were sacrificed for tissues harvesting at 3 days. e Naïve mice were randomly assigned to vehicle or rmC3a treatment; at 6 h after intranasal rmC3a or vehicle administration, mice were sacrificed for tissue harvesting. f Mice were randomly assigned to three groups: sham + vehicle, surgery + vehicle, and surgery + rmC3a. 30 min after rmC3a or vehicle administration, mice were trained for trace fear conditioning. Mice underwent surgery or sham 30 min after training. The context test was performed at 1 day after surgery or sham procedure

Surgery

Orthopedic surgery was performed as previously described [23]. Briefly, mice received an open tibia fracture with intramedullary pinning under 2% isoflurane anesthesia. Buprenorphine (0.1 mg/kg) was administered subcutaneously after anesthesia induction. Sham mice underwent exactly the same anesthesia and analgesia but without surgical intervention.

Intranasal administration of recombinant mouse C3a

Intranasal drug administration was performed as previously described [24] with minor modifications. Mice were acclimated for handling to minimize stress response before drug administration. Recombinant mouse C3a (rmC3a) (10 μg/kg, R&D systems, #8085-C3-025) was dissolved in PBS and intranasally given to each restrained awake mouse in a total volume of 10 μL. Drugs were ejected as small droplets using a pipettor and inhaled through the mouse’s nostril. Vehicle-treated mice were given an equal volume of PBS. Timepoints for intranasal C3a delivery in each experiment were described in Fig. 1d–f. This intranasal approach has been successfully used to deliver exogenous C3a to the mouse brain in previous studies [25, 26].

Trace fear conditioning

Trace fear conditioning has been widely used for assessing hippocampal-dependent memory in PND [4]. 30 min after C3aR antagonist, rmC3a, or vehicle administration, mice received a training session to associate a conditional stimulus (context) with an unconditional stimulus (two periods of 2-s foot-shocks of 0.75 mA each). 30 min after training, mice were subjected to tibia fracture or sham surgery. One or 3 days after training, mice were tested in the same context but received no unconditional stimulus (foot shocks). Freezing behavior for each mouse was recorded and analyzed by a camera-based monitoring system (Xeye Fcs system, Beijing MacroAmbition S&T Development Co., Ltd., Beijing, China).

Statistics

Statistical analysis was performed with GraphPad Prism V6 (GraphPad Software, La Jolla, CA). Comparisons between different groups were made using one-way analysis of variance (ANOVA) with repeated measures followed by Tukey’s or Student-Newman-Keuls test. Unpaired Student’s t test was used for comparisons between two groups. Relationships between two variables were evaluated using linear regression. Statistical significance was indicated when p < 0.05. Data are means ± standard error of the mean.

To illustrate the role of C3/C3aR signaling in surgery-induced neuroinflammation, we examined the effects of a selective C3aR antagonist on pro-inflammatory cytokines, microglial activation, and neutrophil infiltration in the hippocampus.

Hippocampal-dependent memory dysfunction after orthopedic surgery is ameliorated by C3aR blockade. Quantification of the percentage of freezing behavior during the context test on postoperative day 3. Data analyses were performed using one-way analysis of variance followed by Tukey post hoc test. **p < 0.01, ****p < 0.0001

Orthopedic surgery and C3/C3aR signaling in the hippocampus

Under pathological conditions, excessive hippocampal C3 deposition has been implicated in the development of many neurological disorders [13, 14]. In the current model, we found an early and significant elevation of hippocampal C3 levels after orthopedic surgery, supporting the involvement of complement activation in the pathophysiology of PND. Notably, we found C3 was primarily expressed in astrocytes, but not in microglia or neurons. Previous studies have shown that potent inducers of C3 synthesis are IL-1β for astrocytes [30] and tumor necrosis factor-α (TNF-α) for microglia ex vivo [31] while the former but not the latter is elevated in the hippocampus after orthopedic surgery [32, 33]. Thus, astrocytes might be major source of C3 in the current model, which needs further interrogation by future studies.

Microglia are the main cell type that express C3aR in the CNS [19]. Microglial C3aR has been reported to mediate neuroinflammation, β-amyloid pathology, and synapse loss [19, 20]. Here, we showed that C3aR expression in microglia in the hippocampus increased at 1 day after surgery, suggesting orthopedic surgery activates microglial C3aR. These findings also implicate the potential crosstalk between astrocytes and microglia through C3/C3aR signaling. In fact, activated microglia are potent inducers of reactive A1 astrocytes following secretion of pro-inflammatory cytokines like TNF-α, IL-1β, and C1q [34]. This may provide additional targets for upstream modulation of complement signaling and glia activation in PND.

C3aR activation and neuroinflammation

Postoperative neuroinflammation involves elevation of pro-inflammatory cytokines [32, 33], neutrophil infiltration [35], and glia activation [23]. In the hippocampus, pro-inflammatory cytokines are acutely, yet transiently, elevated after surgery and return to baselines by postoperative day 3 [33, 36]. This increase is partially due to local de novo synthesis, with higher mRNA and protein levels of IL-1β and IL-6 in the hippocampus [36]. In a mouse model of Alzheimer’s disease, C3 knockout reduced pro-inflammatory cytokine expressions in the brain, indicating a key role for C3 and/or its downstream signaling in cytokine productions. Here, we found that C3aR blockade also reduced IL-1β and IL-6 levels already at 6 h while C3aR activation by both exogenous C3a and surgical insult prolonged the IL-1β and IL-6 upregulation at 3 days after orthopedic surgery. Of note, activated microglia are one of the primary sources of pro-inflammatory cytokines in the inflamed CNS [37]. Thus, our findings suggest that C3aR activation contributes to hippocampal IL-1β and IL-6 elevations after orthopedic surgery, possibly through microglial activation, although impaired endothelial function with infiltration of peripheral immune cells is also observed following surgery [5, 36, 38]. In fact, neuroinflammation can be also triggered by peripheral factors, including immune cells like macrophages and neutrophil infiltration [39], which is associated with PND [35]. Microgliosis has long been implicated in PND, although the underlying mechanisms for microglial activation remain unclear [3]. We found microglia activation at 1 day after surgery was effectively reduced by pretreatment with C3aR antagonist. Consistent with our finding, previous work showed that C3aR blockade attenuates hippocampal microgliosis in a mouse model of Alzheimer’s disease [19]. Notably, C3aR is expressed in brain endothelial cells [40] and C3/C3aR signaling has been reported to mediate neutrophil infiltration into the brain following lipopolysaccharide administration [41]. Under neuroinflammatory conditions, increased expression of adhesion molecules in activated endothelial cells can mediate the recruitment of neutrophils into the brain parenchyma [42]. Here, we showed that C3aR activation after orthopedic surgery contributes to neutrophil infiltration in the hippocampus, possibly by modifying adhesion molecule expressions in hippocampal endothelium and disrupting BCSFB function.

Complement activation and synapse loss

Microglia-mediated synapse loss has been implicated in the pathophysiology of PND [8]. Upregulation of CD68 immunoreactivity indicates enhanced phagocytic activity in microglia and can be related to increased synapse engulfment [16, 28]. In the current study, we showed a linear relationship between loss of synaptic proteins and CD68 immunoreactivity, suggesting orthopedic surgery increases microglial phagocytosis of hippocampal synapses. Furthermore, surgery-induced microglial CD68 upregulation and synapse loss were both attenuated by C3aR blockade. Previous work investigating the role of complement-microglia axis in synapse elimination demonstrated both C3 and C3aR knockout were protected by synapse loss after West Nile virus infection, implicating a pivotal role for C3/C3aR signaling in microglia-mediated synapse elimination [20]. Although we do not have direct evidence showing surgery-activated microglia to engulf synapses in the present study, our findings provide initial evidence that C3aR activation contributes to synapses loss after orthopedic surgery.

C3aR signaling and choroidal BCSFB dysfunction

The choroid plexus consists of an organized structure of epithelial cells regulating blood-CSF interactions and promoting the clearance of noxious molecules [43]. Choroidal BCSFB dysfunction underlies leukocyte infiltration, reduced neurogenesis, and cognitive decline in many neurological diseases [43]. In the current study, we found orthopedic surgery significantly induced C3 deposition in the choroid plexus. Furthermore, BCSFB permeability, as assessed by IgG deposition, was increased after surgery and normalized by C3aR blockade. Pharmacological activation of C3aR in naïve mice mimicked surgery-induced IgG elevation in the choroidal BCSFB, suggesting C3/C3aR is involved in BCSFB disruption after surgery.

Choroidal BCSFB is a common entry point for leukocyte infiltration into the brain [43] and may allow systemic factors to enter the CNS and contribute to neuroinflammation in PND. Indeed, peripheral neutrophils, macrophages, and T cells can migrate into the brain through BCSFB as shown in animal models of stroke, traumatic brain injury, and Alzheimer’s disease [44]. The choroid plexus also constitutively expresses markers of epithelial cells such as ICAM-1 and VCAM-1 [45]. Upon activation, they mediate leukocyte infiltration into the CNS [46]. Here, we found that ICAM-1 and VCAM-1 were markedly increased in the choroid plexus postoperatively, suggesting surgery activates choroidal epithelium. This surgery-induced epithelial activation may further contribute to neutrophil and macrophage infiltration [5, 36].

Memory deficits and C3 signaling modulation

Orthopedic surgery has been increasingly shown to impair memory processes in rodent models [5, 32, 36, 47], and it commonly affects the recovery of patients after procedures like hip joint replacement [48]. Here, we show that surgery-induced cognitive impairment can be attenuated by prophylactic C3aR blockade and, conversely, we could exacerbate cognition by exogenous C3a administration. The effects of C3aR signaling manipulation on cognition may be due to several factors including changes in neuroinflammation, synapse numbers, and choroidal BCSFB function as reported in this study. Additional cognitive testing may provide insights into the role of complement signaling in PND. Notably, a preliminary clinical trial in cardiac surgical patients found no significant improvement in global cognition following administration of a monoclonal antibody directed against the C5 complement component, although some improvements were observed in the visuo-spatial domain testing [49]. Future studies should further evaluate the components of the complement cascade as well as the timing for possible interventions.

Some limitations of our study must be pointed out. First, we could not exclude the possibility that the therapeutic effects of C3aRa were partially due to its systemic effects as macrophages, which also expressed C3aR [50], are recruited into the hippocampus after surgery to exacerbate neuroinflammation and cognitive dysfunction [5, 36]. Second, further studies are needed to understand the neuro-glia crosstalk in this model and the contribution of astrocytic C3-microglial C3aR signaling on synaptic pruning and postoperative neuroinflammation. Third, we only studied the relatively short-term effects of C3/C3aR signaling manipulations on neuroinflammation and cognition. Although this may inform about the pathogenesis of acute cognitive deficits, like postoperative delirium, longer-lasting assessments combined with more clinically relevant models (i.e., aging, diabetes) should be sought in future studies.

In summary, activation of C3/C3aR signaling after orthopedic surgery contributes to postoperative neuroinflammation, synapse loss, BCSFB dysfunction, and ensuing cognitive impairment. C3aR blockade may represent a promising target for PND and future studies should further evaluate the role of complement signaling after major surgery.

Funding

This work was supported by grant no. 81371199 from the National Natural Science Foundation of China, Beijing, China (to Dr. Wu).

Availability of data and materials

All data and materials related to this study are available upon request from the corresponding author (AW).

Authors’ contributions

CX and AW designed the experiments. CX, JL, DL, and JZ performed the experiments and statistical analyses. CX, NT, and AW wrote the manuscript. All authors read, revised, and approved the final manuscript.

Ethics approval and consent to participate

All procedures were approved by Institutional Animal Care and Use Committee at Capital Medical University (Beijing, China) and carried out under the rules of Medical Research Center of Beijing Chao-Yang Hospital (Beijing, China).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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